This invention relates to a method for maximization of artemisinin production by the plant Artemisia annua belonging to family: Asteraceae. More particularly, the invention is developed through agro-technology involving method of optimizing the planting time, transplanting scheduling, population density, number of harvests and harvesting schedule leading to enhanced yields of artemisinin and related metabolites which have pharmaceutical value of anti-infectives, particularly as antimalarial drug.
The plant Artemisia annua (family: Asteraceae ) is valued for producing a sesquiterpenoid lactone endoperoxide named xe2x80x98artemisininxe2x80x99 which is a promising antimalarial drug and effective against Plasmodium falciparum, P. vivax and drug resistant parasites at nanomolar concentration. The compounds xcex1 and xcex2 arteethers which are synthesized from dihydroartemisinin by etherification with ethanol, were developed as antimalarial drug in India by Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow and Central Drug Research Institute (CDRI), Lucknow after phase III clinical trial. Artemisinins are active against Schistosoma mansoni and S. japonicum, in-vitro and in experimental animal models. These compounds are also active against Leishmania major, Toxoplasma gondii and Pneumocystis carinii in-vitro and against P.carinii in-vivo. Artemisinins have immunosuppressive activity and also potential anticancer activity. Considering the importance of artemisinins which is tedious and difficult to synthesize chemically an all out programme was undertaken to develop Artemisia annua plant varieties with high artemisinin content, development of agrotechnology for increase yield of these compounds, followed by improved extraction procedure. In this direction the inventors were successful in developing and releasing a variety named xe2x80x9cJeevan Rakshaxe2x80x9d from an isolated population containing high artemisinin in the foliage (0.5 to 1.0%) (Sushil Kumar et al. 1999. Journal of Medicinal and Aromatic Plant Sciences. 21:47-48.) This plant xe2x80x9cJeevan Rakshaxe2x80x9d not only produces high artemisinin but also maintains the synchronized conversion to higher level of artemisinin during May to October. As the content of artemisinin fluctuates from zero level at the time of planting to more than 0.4 to 1.00% during May and June with subsequent functions of increase till October, it was necessary to scientifically develop cultivation methodology for the crop to maximize the vigour of the foliage and biosynthesis of artemisinin by systematic scheduling. For this purpose the inventors carried out planned experiments with variation in planting times, population density and number of harvest from the crop to increase the yield from limited area within optimum span of time.
The main object of the invention relates to a method for maximize the artemisinin yield of the plant Artemisia annua by scheduling transplanting time and following a multiple harvesting schedule coinciding with the higher artemisinin accumulation in the plant.
In another object of the invention relates to provide a method for maximizing the yield of artemisinin to the tune of 85-95 kg/ha by following the schedule of the invention.
In yet another object of the invention relates to a method wherein the yield of artemisinin is maximized by maintaining the schedule defined in the invention for different transplanting time of the plant.
To meet the above objects and others, the present invention provides a method for maximizing the artemisinin yield of the plant Artemisia annua by scheduling transplanting time and following a multiple harvesting schedule coinciding with the higher artemisinin accumulation in the plant, wherein the method comprises of the following steps;
(a) sowing seeds of Artemisia annua plant on raised bed nursery during second and third week of December and maintaining the optimum moisture throughout,
(b) transplanting seedlings thus obtained bearing at least 5-15 leaves into the main field fertilized with NPK @ 80,40,40 kg/ha to achieve a population density of 50,000 to 2,00,000 per ha followed by light irrigation in the second week of March and irrigation every fortnight there after,
c) harvesting the crop four times by cutting the plant tops leaving 75-100 cm part of plant for further regeneration, the said harvests are performed in a manner that the first harvest is done in fourth week of May, second harvest in third week of July, third harvest in second week of September and fourth harvest in third week of October of each year, and at each harvesting time care is taken to care at least one green branch, and
d) extracting artemisinin from the plant tissue immediately after each harvest.
It is therefore a intent of the invention to provide the crop of Artemisia annua, a process for increasing the foliage harvest coupled with the maximum artemisinin yield.
The phenotype of a plant is the consequence of interaction between the genotype and the environment. The genotype is the genetic make up which directs the plant to express all characters. But proper expression needs the signal from the environment to express at a particular time, stage and tissue. This is called temporal and spatial expression of genetic traits, which includes synthesis of important chemicals and secondary metabolites like artimisinin in case of Artemisia annua. Based on this fact, it is correct to predict that a plant is not likely to produce and accumulate secondary metabolite just after germination from the seed. Same is true in case of the plant Artemisia annua requiring a certain stage of maturity to start producing artemisinin. Now at the time of production of the compound artemisinin, the plant should have sufficient foliage to accumulate this secondary metabolite. Further, the fluctuating environmental conditions also fluctuate the synthesis of these products. A better genotype responds to these signals in a more vigorous way compared to a weak genotype. At the macro level of commercial crop cultivation in the field environment cannot be manipulated as in the case of cultures in the laboratory. However, critical factors like planting date, number of harvests, harvesting schedules and population density can be manipulated for adjusting to the environmental conditions for maximizing the crop as well as critical chemical production.
With these objective, planned experiments were carried out on the high yielding plant variety xe2x80x9cJeevan Rakshaxe2x80x9d of Artemisia annua, to optimize the agrotechnological process to maximize the yield of herbage and thus artemisinin in a single season so that the method, sequence and optimized scheduling approach can be utilized universally with varying crop seasons in different climatic regions globally by skilled modifications.